Growth as an interconnected mycelial network is characteristic of filamentous fungi and has been subject to scientific investigation since the seminal works of Buller at the start of the 20th century (1–3). We have increasingly detailed understanding of the fundamental cellular processes needed to form a network, such as hyphal tip growth (4), septation (5, 6), hyphal orientation (7), branching (8), and fusion (9–13) (Fig. 1A and C). In contrast, we know far less about the molecular events at the next physical scale that leads to hyphal aggregation and hyphal differentiation, and how these impact physiological processes such as long-distance resource distribution and biomass recycling. For example, while direct uptake and intrahyphal nutrient diffusion may be sufficient to sustain short-range local growth when resources are abundant (14), long-distance translocation is required to deliver nutrients at a sufficient rate to growing tips, particularly in fungi that form large networks on the forest floor that are too large to distribute nutrients through diffusion alone. We know little about the quantitative contribution of different potential transport pathways, such as cytoplasmic streaming, vesicle transport, growth-induced mass flow, or evaporative mass flow, to net fluxes and overall nutrient dynamics, and how they might vary between species and developmental stage (15–17). Nevertheless, the behavior of the growing mycelial network emerges from the interaction of many such processes and requires an integrated view to understand the overall impact on fungal behavior (18–20). Our understanding is further constrained by inferences drawn from a limited number of genetically tractable model filamentous species grown under laboratory conditions (abundant, evenly dispersed, low-molecular-weight resources, high relative humidity, constant light and temperature) compared with real-world conditions (patchy, recalcitrant, ephemeral resources, fluctuating temperature, light and relative humidity).

Mycelial network formation: tip growth and fluid flows. (A) Hyphae grow by extension at the tip through polarized secretion of wall materials from macrovesicles and chitosomes at the apex, choreographed by the Spitzenkörper. Membrane is recycled distal to the tip into early endosomes by endocytosis. Endosomes may also form multivesicular bodies (MVBs) that may be involved in unconventional secretion of exosomes. Transport of secretory vesicles, endosomes, and other organelles along microtubule and microfilament networks also generates cytoplasmic streaming within the apical compartment. Although wall plasticity controls hyphal extension, the driving force involves maintenance of sufficient turgor pressure by water uptake through osmosis in response to accumulation of solutes. Turgor pressure may be sensed and regulated by a mitogen-activated protein kinase (MAPK) cascade or stretch-activated channels, such as mid1. If the site of water uptake required for the volume increase during growth is distal to the tip, growth-induced mass flows will also help to move organelles and solutes toward the tip. Based on references 4, 43, 49, and 64. (B) Water uptake into the hypha occurs to allow tip expansion and is driven by the transverse hydrostatic pressure in response to the difference in concentration of osmotically active solutes between the medium and the hypha. The transverse hydraulic conductivity depends on the permeability of the plasma membrane and aquaporins (AQPs) in parallel, and the wall and other surface layers, such as hydrophobins, in series. Longitudinal flow in the lumen of the hypha is lamina and follows Poiseuille flow. Based on references 32, 33, 49, and 79. (C) Variation in septal pore structure in different fungal taxa. Redrawn from reference 96 with permission. (D) Impact of the septa and septal pores on fluid flows. The change in cross-sectional area causes an increase in velocity by several orders of magnitude and also increases the wall shear stress within the pore. Flow may deviate from parabolic profile expected from the Hagen-Poiseuille equation because of the density of organelles in the cytoplasm. In addition, there may be eddy currents near the pore opening that trap nuclei, vacuoles, and other organelles. Based on references 32, 65, 68, and 69.

10.1128/9781555819583/fig15-1_thmb.gif

10.1128/9781555819583/fig15-1.gif

Figure 1

Mycelial network formation: tip growth and fluid flows. (A) Hyphae grow by extension at the tip through polarized secretion of wall materials from macrovesicles and chitosomes at the apex, choreographed by the Spitzenkörper. Membrane is recycled distal to the tip into early endosomes by endocytosis. Endosomes may also form multivesicular bodies (MVBs) that may be involved in unconventional secretion of exosomes. Transport of secretory vesicles, endosomes, and other organelles along microtubule and microfilament networks also generates cytoplasmic streaming within the apical compartment. Although wall plasticity controls hyphal extension, the driving force involves maintenance of sufficient turgor pressure by water uptake through osmosis in response to accumulation of solutes. Turgor pressure may be sensed and regulated by a mitogen-activated protein kinase (MAPK) cascade or stretch-activated channels, such as mid1. If the site of water uptake required for the volume increase during growth is distal to the tip, growth-induced mass flows will also help to move organelles and solutes toward the tip. Based on references 4, 43, 49, and 64. (B) Water uptake into the hypha occurs to allow tip expansion and is driven by the transverse hydrostatic pressure in response to the difference in concentration of osmotically active solutes between the medium and the hypha. The transverse hydraulic conductivity depends on the permeability of the plasma membrane and aquaporins (AQPs) in parallel, and the wall and other surface layers, such as hydrophobins, in series. Longitudinal flow in the lumen of the hypha is lamina and follows Poiseuille flow. Based on references 32, 33, 49, and 79. (C) Variation in septal pore structure in different fungal taxa. Redrawn from reference 96 with permission. (D) Impact of the septa and septal pores on fluid flows. The change in cross-sectional area causes an increase in velocity by several orders of magnitude and also increases the wall shear stress within the pore. Flow may deviate from parabolic profile expected from the Hagen-Poiseuille equation because of the density of organelles in the cytoplasm. In addition, there may be eddy currents near the pore opening that trap nuclei, vacuoles, and other organelles. Based on references 32, 65, 68, and 69.

Mycelial network formation: branching, fusion, and multihyphal aggregate formation. (A) Hyphae may branch subapically or by tip splitting to explore the substrate or to form aerial hyphae that are often insulated by hydrophobins. At the colony margin, tips avoid each other, while secondary branch hyphae in the colony interior can show positive autotropism and fuse by anastomosis. (B) The velocity of mass flow within the network depends critically on the site of water uptake for growth. If all water uptake is distal from the tips, the velocity scales in proportion to the number of downstream tips. If uptake is equal everywhere, the flow rate is constant at the speed of hyphal extension, while if uptake is solely at the tips, there is no long-distance movement. Based on references 32 and 33. (C) Schematic representation of the formation of strands, cords, and rhizomorphs, showing progressive differentiation of vessel hyphae as potential conduits for long-distance transport. Scale bars are approximate. Based on references 127, 129, 134, 228, and 265. (D) Schematic representation of how circulating fluid flows might operate within a hyphal cord. Acropetal mass flows would take place in the vessel hyphae in response to growth, evaporation, or exudation at the tips, while basipetal flows take place simultaneously through cytoplasmic hyphae by cytoplasmic streaming. Redrawn from reference 17 with permission.

10.1128/9781555819583/fig15-2_thmb.gif

10.1128/9781555819583/fig15-2.gif

Figure 2

Mycelial network formation: branching, fusion, and multihyphal aggregate formation. (A) Hyphae may branch subapically or by tip splitting to explore the substrate or to form aerial hyphae that are often insulated by hydrophobins. At the colony margin, tips avoid each other, while secondary branch hyphae in the colony interior can show positive autotropism and fuse by anastomosis. (B) The velocity of mass flow within the network depends critically on the site of water uptake for growth. If all water uptake is distal from the tips, the velocity scales in proportion to the number of downstream tips. If uptake is equal everywhere, the flow rate is constant at the speed of hyphal extension, while if uptake is solely at the tips, there is no long-distance movement. Based on references 32 and 33. (C) Schematic representation of the formation of strands, cords, and rhizomorphs, showing progressive differentiation of vessel hyphae as potential conduits for long-distance transport. Scale bars are approximate. Based on references 127, 129, 134, 228, and 265. (D) Schematic representation of how circulating fluid flows might operate within a hyphal cord. Acropetal mass flows would take place in the vessel hyphae in response to growth, evaporation, or exudation at the tips, while basipetal flows take place simultaneously through cytoplasmic hyphae by cytoplasmic streaming. Redrawn from reference 17 with permission.

Growth-induced mass flows explain long-distance nutrient movement in Phanerochaete velutina. (A) Structure of the mycelial network after 21 days growing from a wood resource across compressed black sand. (B) The network architecture is extracted using intensity-independent, phase-congruency tensors and watershed segmentation from experimental time series. The output is a set of weighted adjacency matrices of the length, width, and volume of each cord. In this image, the cord width is pseudo-color coded from 50 μm (blue) to 500 μm (red). (C) The network structure and growth are input into a biophysical advection/diffusion/delivery (ADD) model, using growth-induced mass flow to predict the pattern of resource translocation. The predicted amount of radiolabel is color coded on a rainbow scale from blue (zero) to red (maximum). (D) The actual pattern of nutrient movement is then determined experimentally by using the nonmetabolized amino acid analogue 14C-amino isobutyrate (14C-AIB) and photon-counting scintillation imaging (PCSI). The amount of 14C-AIB is color coded on a rainbow scale from blue (low) to red (high). The ADD model of growth-induced mass flow predicts the distribution of radiotracer in a complex network of fungal cords with a Pearson correlation coefficient 0.56. From Heaton et al. (32) with permission.

10.1128/9781555819583/fig15-3_thmb.gif

10.1128/9781555819583/fig15-3.gif

Figure 3

Growth-induced mass flows explain long-distance nutrient movement in Phanerochaete velutina. (A) Structure of the mycelial network after 21 days growing from a wood resource across compressed black sand. (B) The network architecture is extracted using intensity-independent, phase-congruency tensors and watershed segmentation from experimental time series. The output is a set of weighted adjacency matrices of the length, width, and volume of each cord. In this image, the cord width is pseudo-color coded from 50 μm (blue) to 500 μm (red). (C) The network structure and growth are input into a biophysical advection/diffusion/delivery (ADD) model, using growth-induced mass flow to predict the pattern of resource translocation. The predicted amount of radiolabel is color coded on a rainbow scale from blue (zero) to red (maximum). (D) The actual pattern of nutrient movement is then determined experimentally by using the nonmetabolized amino acid analogue 14C-amino isobutyrate (14C-AIB) and photon-counting scintillation imaging (PCSI). The amount of 14C-AIB is color coded on a rainbow scale from blue (low) to red (high). The ADD model of growth-induced mass flow predicts the distribution of radiotracer in a complex network of fungal cords with a Pearson correlation coefficient 0.56. From Heaton et al. (32) with permission.

Mycelial networks in woodland. (A) Map of a mycelial cord network of Phanerochaete velutina revealed by carefully removing surface litter layer, recovering, and then re-revealing 13 months later. From Thompson and Rayner (155) with permission. (B) Network of Megacollybia platyphylla on the floor of a mixed deciduous woodland, following removal of surface litter.

10.1128/9781555819583/fig15-4_thmb.gif

10.1128/9781555819583/fig15-4.gif

Figure 4

Mycelial networks in woodland. (A) Map of a mycelial cord network of Phanerochaete velutina revealed by carefully removing surface litter layer, recovering, and then re-revealing 13 months later. From Thompson and Rayner (155) with permission. (B) Network of Megacollybia platyphylla on the floor of a mixed deciduous woodland, following removal of surface litter.

Effects of resource addition and grazing on mycelial networks. Mycelial cord systems (99 days old) of Phanerochaete velutina growing from centrally positioned beech wood inocula across the surface of compressed nonsterile soil in 50 × 50 cm trays. (A) With four additional beech wood blocks added behind the mycelial margin 36 days after central inoculation. (B) With no additional resources added. (C) As in A, but with 250 laboratory-reared collembola, Folsomia candida, added 49 days after the central inoculums. (D) As in C, but with no additional resources. From Wood et al. (170), with permission.

10.1128/9781555819583/fig15-5_thmb.gif

10.1128/9781555819583/fig15-5.gif

Figure 5

Effects of resource addition and grazing on mycelial networks. Mycelial cord systems (99 days old) of Phanerochaete velutina growing from centrally positioned beech wood inocula across the surface of compressed nonsterile soil in 50 × 50 cm trays. (A) With four additional beech wood blocks added behind the mycelial margin 36 days after central inoculation. (B) With no additional resources added. (C) As in A, but with 250 laboratory-reared collembola, Folsomia candida, added 49 days after the central inoculums. (D) As in C, but with no additional resources. From Wood et al. (170), with permission.

Macroscopic network analysis of Phallus impudicus growing on compressed soil. (A) Original image of the mycelial network after 21 days. (B) Network architecture automatically extracted using phase-congruency edge enhancement, watershed segmentation, and link pruning to give a single-pixel-wide skeleton pseudo-color coded by the cord width. (C) Conversion to a graph representation whereby each node (junction or tip) is connected by edges that are pseudo-color coded by the average width of the cord segment from 50 μm (blue) to 500 μm (red). The structure of the network within the wood resource cannot be defined so any cord incident on the boundary is connected to a central node with a link set to the maximum width. (D) Characterization of the cord betweenness centrality as a measure of how important each cord is to transport through the network from the resource to every other node, with the color code ranging from blue (low importance) to red (high importance).

10.1128/9781555819583/fig15-7_thmb.gif

10.1128/9781555819583/fig15-7.gif

Figure 7

Macroscopic network analysis of Phallus impudicus growing on compressed soil. (A) Original image of the mycelial network after 21 days. (B) Network architecture automatically extracted using phase-congruency edge enhancement, watershed segmentation, and link pruning to give a single-pixel-wide skeleton pseudo-color coded by the cord width. (C) Conversion to a graph representation whereby each node (junction or tip) is connected by edges that are pseudo-color coded by the average width of the cord segment from 50 μm (blue) to 500 μm (red). The structure of the network within the wood resource cannot be defined so any cord incident on the boundary is connected to a central node with a link set to the maximum width. (D) Characterization of the cord betweenness centrality as a measure of how important each cord is to transport through the network from the resource to every other node, with the color code ranging from blue (low importance) to red (high importance).

Network taxonomy. Taxonomies of 270 fungal (and slime mold) networks based on community structure using modularity optimization with path score (PS) values as the edge weights (205). The dendrogram was produced from the mesoscopic structure of each network as an indication of how similar different networks are to each other. The species abbreviations are coded as follows: Pp, Physarum polycephalum; Pv, Phanerochaete velutina; Ag, Agrocybe gibberosa; Pi, Phallus impudicus; Rb, Resinicium bicolor; and Sc, Strophularia caerulea. The levels of resources and amount of grazing are color coded from low (blue) to high (red). Substrate is coded as blue for agar, white for sand, and red for compressed, nonsterile soil. Interactions are coded as blue for no interaction, or red grown in competition with Hypholoma fasciculare. At the bottom of the figure, the logarithms of number of nodes N, number of edges M, and the edge density ρ = 2M/N(N − 1) are also shown. From reference 205 with permission.

10.1128/9781555819583/fig15-8_thmb.gif

10.1128/9781555819583/fig15-8.gif

Figure 8

Network taxonomy. Taxonomies of 270 fungal (and slime mold) networks based on community structure using modularity optimization with path score (PS) values as the edge weights (205). The dendrogram was produced from the mesoscopic structure of each network as an indication of how similar different networks are to each other. The species abbreviations are coded as follows: Pp, Physarum polycephalum; Pv, Phanerochaete velutina; Ag, Agrocybe gibberosa; Pi, Phallus impudicus; Rb, Resinicium bicolor; and Sc, Strophularia caerulea. The levels of resources and amount of grazing are color coded from low (blue) to high (red). Substrate is coded as blue for agar, white for sand, and red for compressed, nonsterile soil. Interactions are coded as blue for no interaction, or red grown in competition with Hypholoma fasciculare. At the bottom of the figure, the logarithms of number of nodes N, number of edges M, and the edge density ρ = 2M/N(N − 1) are also shown. From reference 205 with permission.

In silico evaluation of network robustness. Networks of Phanerochaete velutina (red) and Phallus impudicus (green) were attacked in silico by progressively removing links in the network at random, and calculating the number of paths in the network that still remain. In each case, five examples are shown. Note that the P. velutina network breaks down more rapidly than the P. impudicus. From D. A’Bear, L. Boddy, and M. D. Fricker, unpublished data.

10.1128/9781555819583/fig15-9_thmb.gif

10.1128/9781555819583/fig15-9.gif

Figure 9

In silico evaluation of network robustness. Networks of Phanerochaete velutina (red) and Phallus impudicus (green) were attacked in silico by progressively removing links in the network at random, and calculating the number of paths in the network that still remain. In each case, five examples are shown. Note that the P. velutina network breaks down more rapidly than the P. impudicus. From D. A’Bear, L. Boddy, and M. D. Fricker, unpublished data.

Network analysis of the ectomycorrhizal fungus Paxillus on pine. (A)Pinus seedling infected with Paxillus. The dotted white circle marks the loading site for radioactive 14C-AIB. The dotted red lines indicate the routes for preferential transport of isotope to the roots from C. (B) Automated extraction of both the plant root and extra-radical mycorrhizal network using phase-congruency enhancement, watershed segmentation, and cord width measurement. (C) Scintillation image of nutrient movement along selected pathways from the fungus to the plant root. From R. Tajuddin, D. Johnson, and M.D. Fricker, unpublished data.

10.1128/9781555819583/fig15-10_thmb.gif

10.1128/9781555819583/fig15-10.gif

Figure 10

Network analysis of the ectomycorrhizal fungus Paxillus on pine. (A)Pinus seedling infected with Paxillus. The dotted white circle marks the loading site for radioactive 14C-AIB. The dotted red lines indicate the routes for preferential transport of isotope to the roots from C. (B) Automated extraction of both the plant root and extra-radical mycorrhizal network using phase-congruency enhancement, watershed segmentation, and cord width measurement. (C) Scintillation image of nutrient movement along selected pathways from the fungus to the plant root. From R. Tajuddin, D. Johnson, and M.D. Fricker, unpublished data.

41.SteinbergG.2007. Hyphal growth: a tale of motors, lipids, and the Spitzenkörper. Eukaryot Cell6:351–360.[PubMed]

42.BalmantW,, Sugai-GuériosMH,, CoradinJH,, KriegerN,, Furigo JuniorA,, MitchellDA.2015. A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae. PLoS One10:e0120307.[PubMed]

51.OlssonS,, JenningsDH.1991. A glass-fiber filter technique for studying nutrient-uptake by fungi: the technique used on colonies grown on nutrient gradients of carbon and phosphorus. Exp Mycol15:292–301.

52.OlssonS,, JenningsDH.1991. Evidence for diffusion being the mechanism of translocation in the hyphae of three molds. Exp Mycol15:302–309.

156.SmithML,, BruhnJN,, AndersonJB.1992. The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature356:428–431.

157.FergusonBA,, DreisbachTA,, ParksCG,, FilipGM,, SchmittCL.2003. Coarse-scale population structure of pathogenic Armillaria species in a mixed-conifer forest in the Blue Mountains of northeast Oregon. Can J Res33:612–623.

164.AgererR.2001. Exploration types of ectomycorrhizae: a proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza11:107–114.

185.NopharatanaM,, MitchellDA,, HowesT.2003. Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface. Biotechnol Bioeng84:71–77.[PubMed]

186.NopharatanaM,, MitchellDA,, HowesT.2003. Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system. Biotechnol Bioeng81:438–447.[PubMed]